Abstract
Tension applied to the plasma membrane (PM) is a global mechanical parameter involved in cell migration. However, how membrane tension regulates actin assembly is unknown. Here, we demonstrate that FBP17, a membrane-bending protein and an activator of WASP/N-WASP-dependent actin nucleation, is a PM tension sensor involved in leading edge formation. In migrating cells, FBP17 localizes to short membrane invaginations at the leading edge, while diminishing from the cell rear in response to PM tension increase. Conversely, following reduced PM tension, FBP17 dots randomly distribute throughout the cell, correlating with loss of polarized actin assembly on PM tension reduction. Actin protrusive force is required for the polarized accumulation, indicating a role for FBP17-mediated activation of WASP/N-WASP in PM tension generation. In vitro experiments show that FBP17 membrane-bending activity depends on liposomal membrane tension. Thus, FBP17 is the local activator of actin polymerization that is inhibited by PM tension in the feedback loop that regulates cell migration.
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Acknowledgements
We would like to thank T. Inoue for discussions, M. Ichikawa for technical assistance and H. Yamamoto for help with plasmid constructions. This study was supported by a Grant-in-Aid for Scientific Research (C) from the Japan Society for the Promotion of Science (JSPS) to K.T. and T.I., a Grant-in-Aid for Scientific Research (S) from JSPS to T.T. and a Uehara Memorial Foundation grant to K.T.
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K.T. and T.I. conceived the research project. K.T. and T.I. designed and carried out experiments. T.T. provided key reagents. K.T. and T.I. analysed the data and wrote the manuscript.
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Supplementary Figure 2 PM tension sensitive localisation of FBP17.
(a) Tether force measurement of COS-1 cells. Tether force of identical cell was measured before and after hypotonic shock (upper) or hypertonic shock (lower). Values are presented as ratio relative to before treatment. Mean and s.d. are derived from pooled data from three independent experiments. In total, n = 9 events (hypotonic) and n = 8 events (hypertonic) were analysed, respectively. ∗∗P = 0.014,∗P = 0.0012, Student’s t-test. (b) Confocal images of endogenous FBP17, endogenous clathrin and F-actin in COS-1 cell on hypotonic shock. Scale bar, 20 μm. (c) Confocal images of endogenous FBP17 and F-actin (stained with Alexa Fluor 568-conjugated phalloidin) in Swiss 3T3 cells on PDGF stimulation.
Supplementary Figure 3 Manipulation of PM tension affects the localisation of FBP17.
Time-lapse images of COS-1 cell co-expressing GFP-FBP17 and Lifeact-mCherry on addition of deoxycholic acid (400 μM).
Supplementary Figure 4 Models of PM tension fluctuation at the leading edge.
PM at the cell front is pushed outwardly by polymerised actin (blue). When the front membrane retracts backward (a) or curls up by ruffling (b), there emerges a local PM area with reduced tension. The drop in local PM tension induces membrane invaginations by FBP17 (red), which promotes actin assembly. FBP17-mediated actin polymerisation again increases PM tension, resulting in its dissociation from the membrane.
Supplementary Figure 5 Confirmation of effects by RNAi on the expression levels of target proteins.
(a,b) Protein levels in COS-1 cells transfected with the indicated siRNA were analysed by western blot using specific antibodies against FBP17 (a) and N-WASP (b). (c,d) Expressions of Toca family proteins in COS-1 or MDA-MB-231 cells analysed by RT-PCR. Triple RNAi indicates the combination of siRNAs of FBP17, CIP4, and Toca-1.
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Polarisation of FBP17 is induced by PM tension increase.
COS-1cell co-expressing GFP-FBP17 and Lifeact-mCherry was observed by time-lapse microscopy on hypotonic buffer. The movie was taken at 1 frame per 5 s and played at 15 fps. (MOV 14507 kb)
Polarisation of FBP17 is disrupted by PM tension decrease.
COS-1 cell co-expressing GFP-FBP17 and Lifeact-mCherry was observed by time-lapse microscopy on addition of hypertonic buffer. The movie was taken at 1 frame per 5 s and played at 15 fps. (MOV 22709 kb)
Polarisation of FBP17 is induced by PtdIns(4,5)P2 liberation.
COS-1 cell co-expressing GFP-FBP17, CFP-FKBP-PLC δ1 PH domain, and mRFP-FRB-MoA was observed by time-lapse microscopy on addition of rapamycin. The movie was taken at 1 frame per 5 s and played at 15 fps. (MOV 7448 kb)
Polarisation of FBP17 is disrupted by PtdIns(4,5)P2 depletion.
COS-1 cell co-expressing GFP-FBP17, CFP-PM-anchored FRB domain, and mRFP-FKBP-5-phosphatase domain was observed by time-lapse microscopy on addition of rapamycin. The movie was taken at 1 frame per 5 s and played at 15 fps. (MOV 10733 kb)
Dynamics of FBP17 at the leading edge.
COS-1 cell co-expressing GFP-FBP17 and Lifeact-mCherry was observed by time-lapse microscopy. The movie was taken at 1 frame per 5 s and played at 15 fps. (MOV 15287 kb)
Acute disruption of FBP17 polarity by N-WASP inhibition.
COS-1 cell co-expressing GFP-FBP17 and Lifeact-mCherry was observed by time-lapse microscopy on addition of wiskostatin. The movie was taken at 1 frame per 10 s and played at 15 fps. (MOV 3757 kb)
Acute disruption of FBP17 polarity by Arp2/3 complex inhibition.
COS-1 cell co-expressing GFP-FBP17 and Lifeact-mCherry was observed by time-lapse microscopy on addition of CK-666. The movie was taken at 1 frame per 10 s and played at 15 fps. (MOV 8851 kb)
PM tension dependent recruitment of FBP17.
COS-1 cell co-expressing GFP-FBP17 and Lifeact-mCherry was observed by time-lapse microscopy on addition of blebbistatin followed by the treatment of hypertonic buffer. The movie was taken at 1 frame per 5 s and played at 15 fps. (MOV 7257 kb)
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Tsujita, K., Takenawa, T. & Itoh, T. Feedback regulation between plasma membrane tension and membrane-bending proteins organizes cell polarity during leading edge formation. Nat Cell Biol 17, 749–758 (2015). https://doi.org/10.1038/ncb3162
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DOI: https://doi.org/10.1038/ncb3162
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